Asymptomatic pregnant women (from 17 to 37 years old) with gestational age from 12 to 34 weeks were enrolled (n=103) during their prenatal examination in the Obstetrics Service of the Maternal and Child Department of the Prof. A. Posadas National Hospital (Buenos Aires, Argentina) between January and March 2019. Endocervical swab samples were collected under direct visualization during a speculum examination. Swabs were transported in sterile tubes containing Cary Blair transport medium (Britania, Argentina) and stored at 4-8°C until used.

Endocervical swabs were enriched in 3 mL of Tryptic Soy Broth (Oxoid, UK) overnight (ON) at 37°C and shaken at 150 rpm. Then, cultures were streaked into Sorbitol MacConkey (SMAC) agar (Oxoid, UK) and incubated ON at 37°C. SMAC agar was used for a preliminary selection of Enterobacteria based on a differential sorbitol and lactose fermentation profile. SMAC agar is recommended as a selective and differential medium for the detection of STEC O157:H7. Enterobacteria capable of fermenting sorbitol form pink colonies on SMAC agar, while non-sorbitol fermenting bacteria, such as STEC O157:H7, form white/colorless colonies.

Total genomic DNA from colonies grown in SMAC agar was obtained to detect the presence of E. coli by PCR. DNA was purified using PURO bacteria Kit (PB-L products, Bio-Logicos, Argentina) according to the manufacturer’s instructions and quantified by Nanodrop One (Thermo Fisher Scientific, USA). Specific genes encoding for the enzyme beta-glucuronidase (uidA gene) (28) and external membrane protein of E. coli (yaiO gene) (29), and genes specific for portions of the rfb (O-antigen-encoding) regions of STEC O157 (rfbO₁₅₇gene) (11) were detected by PCR using specific primers ( Table 1 ).

PCRs were performed in a total volume of 20 µL: 6 µL H₂O, 3 µL DNA, 0.5 µL of each forward and reverse primer (10 µM) and 10 µL Master Mix 2X Kit (M024, Inbio Highway, Argentina). The thermocycler (Thermo Fisher Scientific, USA) program used consisted of one cycle at 95°C for 10 min followed by 35 cycles of 94°C for 40 s, 58°C for 30 s, and 72°C for 60 s, ending with one cycle of 72°C for 2 min. A total of 10 μL of each PCR reaction product was loaded into an agarose gel to confirm the presence and size of the corresponding amplicon.

Samples positive for yaiO were analyzed for rfbO157 as well as for the presence of other STEC virulence factor genes, including: stx1, stx2, stx2a, stx2c (4), subAB (30), eae (31), lpfAO₁₁₃ (12), hcp (13), iha (32) and sab (15) by PCR ( Table 2 ).

The Vero cell line (Vero), derived from African green monkey kidney, was purchased from the American Type Culture Collection (ATCC-CCL-81, Manassas, VA, USA). The HeLa cell line (ATCC-CCL-2), derived from a human cervix adenocarcinoma, was used as a human endocervical model. The Swan 71 cell line, derived by telomerase-mediated transformation of a 7-week cytotrophoblast isolate, was kindly provided by Dr. Gil Mor, Yale University, New Haven, CT, USA (33).

All cell lines were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12 medium, Sigma Aldrich, USA) supplemented with 10% Fetal Bovine Serum (FBS, Internegocios S.A., Argentina), 100 U/mL penicillin/streptomycin, and 2 mM L-glutamine (GIBCO, USA), and grown at 37°C in a humidified 5% CO₂ incubator. For growth-arrested conditions, the medium was used without FBS.

Endocervical samples that were positive for stx2 by PCR were grown ON in Luria-Bertani Broth (LB) (Sigma Aldrich, USA) at 37°C with shaking at 150 rpm. Then, the cultures were diluted (1:20) in DMEM-F12 medium (Sigma Aldrich, USA), supplemented with 1 mM of HEPES buffer solution (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)) (GIBCO, USA) and grown until exponential phase (OD₆₀₀ = 0.4-0.5) at 37°C with shaking at 150 rpm. Subsequently, bacterial supernatants were collected after centrifugation at 10,000 x g for 5 min and filtered (0.22-μm pore-size filter; Millipore, USA). In some experiments, to induce Stx2 production, mitomycin-C (1 µg/mL) was added at the exponential phase and the bacterial culture was grown for additional 3 h (34).

Cell viability assays were performed as previously described (35). Briefly, 15x 10³ cells/well (Vero, Swan 71, and HeLa) were seeded in 96-well plates and grown at 80% of confluence. Then, cells were exposed for 72 h to different concentrations (1x10^-6 – 0.1 µg/mL) of purified Stx2 (Phoenix Laboratory, Tufts Medical Center, Boston, USA) or to serial dilutions of bacterial supernatant (10^-1-10^-6) in growth-arrested conditions. After treatment, cells were incubated with a neutral red solution (50 µg/mL) at 37°C for 1 h in 5% CO₂. Then, cells were washed and fixed with 1% CaCl₂-1% formaldehyde and finally lysed with 1% acetic acid in 50% ethanol solution to solubilize the neutral red uptake by cells. Absorbance in each well was read at 540 nm in an automated plate spectrophotometer (RT-6000, Rayto Life and Analytical Sciences Co. Ltd., China). Results are expressed as percentage of cell viability, where 100% represents control cells without toxin or supernatant treatment. The 50% cytotoxic dose (CD₅₀) of Stx2 and the bacterial supernatant corresponded to the dilution required to kill 50% of cells.

For the neutralization assay, bacterial supernatant in a dilution required to kill 50% of Vero cells (CD₅₀) was co-incubated with 25 µg/mL of the mouse monoclonal antibody 2E11 against the A-subunit of Stx2 (anti-Stx2 mAb) at 37°C for 1 h (36). The mixture was added to a 96-well culture plate containing Vero cells grown at 80% of confluence and then incubated for 72 h. The cytotoxicity was analyzed by neutral red uptake as previously described. The CD₅₀ of purified Stx2 (1 ng/mL) was used as control.

The enriched bacterial culture from endocervical samples carrying the stx2 gene and showing cytotoxic effects on Vero was streaked onto LB agar plates and incubated ON at 37°C. Grown colonies were picked and added in 96-well plates and grown in LB medium ON at 37°C with agitation at 150 rpm. The next day, wells of each column were pooled and the DNA was purified to test the presence of the stx2 gene. Afterward, individual colonies grown in each well of stx2-positive columns were directly picked and confirmed to be STEC by testing the presence of the stx2 gene by PCR and by Vero cell assays. Isolates were then sub-cultured for further genetic or phenotypic typing.

The serotypes (O and H) of STEC isolates were determined by microagglutination, as described by Blanco etal. (37).

This study had the approval of the Human Research Ethics Committee of the Prof. A. Posadas National Hospital, Buenos Aires, Argentina (Ref # 423 EMnPeSe/20), in accordance with the Argentine Good Clinical Practice Guidelines. All pregnant women were thoroughly informed about the purpose of the study and provided a written informed consent. The exclusion criteria were: women with genital papilloma virus infection (HPV), human immunodeficiency virus (HIV), pelvic inflammatory disease (PID) and sexually transmitted diseases such as chlamydia, gonorrhea, and genital herpes.

Data were plotted and statistically analyzed using Graph Pad Prism 5.0 (San Diego, CA, USA). Cytotoxicity curves were fitted using a four-parameter logarithmic regression. Statistical significance for all experiments was assessed using analysis of variance (ANOVA) with Tukey’s multiple comparison test as a posteriori test. In all cases, statistical significance was set at *p < 0.05.

Sixteen out of the 103 endocervical swab samples developed colonies on SMAC agar. Only non-sorbitol fermenting colonies were observed. Fifteen out of those sixteen samples had the uidA gene and 12 out of the 16 had the yaiO gene ( Table 3 ). Considering that the yaiO gene is more specific than the uidA gene for the identification of E. coli, only those containing the yaiO gene were considered positive for E. coli (29). One sample (S12) was negative for both genes ( Table 3 ). Therefore, 12 out of the 103 endocervical samples recruited for this study (11.7%) were considered positive for E. coli spp. in the endocervical microbiota. The absence of sorbitol-fermenting colonies (characteristic of O157:H7 E. coli) on SMAC agar was confirmed by PCR (none of the samples evaluated were positive for the rfb_O157 gene) ( Table 3 ).

The presence of STEC virulence factors was analyzed in the 12 endocervical samples that developed colonies on SMAC agar and were confirmed positive for E. coli by the presence of the yaiO gene. Seven out of the 12 samples (58.3%) amplified for the stx2 gene, 5 out of the 12 (41.7%) for the lpf_AO113 gene, 8 out of the 12 (66.7%) for the hcpA gene and 3 out of the 12 (25%) for the iha gene ( Table 4 ). None of them were positive for the eae, stx1 or sab genes.

The cytotoxic activity of filter-sterilized bacterial supernatants obtained from endocervical samples positive for E. coli carrying the stx2 gene was evaluated in Vero cells ( Figure 1 ).

Incubation of Vero cells for 72 h with serial dilutions of the bacterial supernatant corresponding to sample 14 (S14-SN) caused a significant cytotoxicity in a dose-dependent manner, reaching a CD₅₀ at 2x10^-4 dilution. The CD₅₀ of Stx2 in S14-SN was equivalent to that elicited by approximately 5 µg/mL of purified Stx2 ( Figure 1 ). In contrast, the other filter-sterilized bacterial supernatants showed no cytotoxic effects on the viability of Vero cells, even if previously grown with mitomycin-C, an inductor of stx2 phages (data not shown).

Neutralization studies were used to confirm that the significant decrease in Vero cell viability caused by S14-SN was due to the cytotoxic action of Stx2. The results showed that S14-SN cytotoxicity was prevented by preincubation of the bacterial S14-SN (1:5000) with the anti-Stx2 mAb (25 µg/mL) at 37°C for 1 h. Neutralization of purified Stx2 was used as control ( Figure 2 ).

A significant cytotoxic effect was observed when monolayers of human endocervical (HeLa) cells were exposed to different concentrations of Stx2 or serial dilutions of the filter-sterilized bacterial supernatants or bacteria isolated from endocervical sample S14 (S14-SN) for 72 h. The CD₅₀ was obtained with 10 ng/mL of purified Stx2 and with a 5x10^-3 dilution of S14-SN. In agreement with the results described above, cytotoxicity was also observed with purified Stx2 on monolayers of extravillous trophoblasts (Swan 71 cells). The CD₅₀ after 72 h of incubation with purified Stx2 was obtained at 100 ng/mL and with a dilution of approximately 2x10^-2 of S14-SN ( Figure 3 ). These results indicate that filter-sterilized bacterial supernatants from the STEC-positive endocervical microbiota can impair endocervical and trophoblast cell viabilities mediated by Stx2.

The bacterial isolate from endocervical sample S14, named STEC 123/21, was subtyped for stx2 by PCR, using specific primers for the stx2a and stx2c genes ( Table 2 ). Figure 4 shows a representative gel of the STEC isolate subjected to PCR assay. Results showed PCR products of the expected sizes, consistent with the presence of the stx2a gene ( Figure 4 ) and the absence of the subAB gene (S14 in Table 3 ).

STEC strain 123/21 belonged to the O113 type, but the H type could not be determined (nondetermined, NT). The analysis of the supernatant from STEC O113:NT demonstrated that it was able to induce a marked decrease in cell viability. The cytotoxicity of the supernatant of this STEC isolate was similar to that obtained from the initial endocervical sample S14 containing other bacteria (SN-S14) ( Figure 5 ).